Fluorescence activated cell sorting (FACS) is a powerful method that has been used to identify cells having a particular phenotype. See Herzenberg et al. (Clincal Chem. 48(10):1819–1827 (2002)) for a review. In some forms, the FACS method has been used in combination with monoclonal antibodies as a reagent to detect cells as having a particular antigen, which is usually indicative of an expressed protein. The method has been used extensively in relation to antigens expressed on the surface of cells, including cells that remain alive during, and after, FACS. Similarly, the method has been used with intracellular reporter gene systems based on the expression of a detectably labeled gene product by the cell.
The method has also been used to detect contents within fixed cells via an alternative form of intracellular FACS. This method permits the detection of intracellular molecules that are not expressed in a labeled form by first fixing the cells followed by permeabilization of the cells to permit entry of a reagent, like an antibody, that binds an intracellular factor. Of course the use of fixation kills all cells in a sample. Thus this intracellular FACS method does not permit the separate identification of viable cells that contain an intracellular molecule from dead, or dying, cells containing the same molecule. This is despite a variety of means and methods known in the field to determine cell viability.
Approaches to determine cell viability are based on the principle of viable cells being capable of excluding certain agents, such as trypan blue and ethidium monoazide. Trypan blue staining for example, is based on cell membrane integrity, which is utilized based upon a presumed correlation to cell death, which permits entry of the dye. Other approaches are based on the principle of viable cells taking up reagents or factors that can be used to identify the cells as having been alive. Examples of this approach include the uptake of radioactive substances, such as tritium-labeled thymidine, or the uptake of a tetrazolium salt, such as the yellow tetrazolium salt MTT, which is enzymatically reduced by dehydrogenases to form insoluble purple formazan crystals by the mitochondria in metabolically active cells. The crystals are solubilized by the addition of an organic solvent, such as isopropyl alcohol or dimethyl sulfoxide, to permit color detection by spectrophotometric means. Unfortunately, the solvents used in a MTT based assay also lyse the cells to result in an overall amount of color to determine the number of viable cells.
References discussing the above methods include van de Loosdrecht, A. A., et al. J. Immunol. Methods 174: 311–320, 1994; Ohno, M., and T. Abe. J. Immunol. Methods 145:199–203, 1991; Ferrari, M., et al. J. Immunol. Methods 131: 165–172, 1990; Alley, M. C., et al. Cancer Res. 48: 589–601, 1988; Carmichael, J., et al. Cancer Res. 47:936–42, 1987; Gerlier, D., and N. Thomasset. J. Immunol. Methods 94: 57–63, 1986; and Mosmann, T. J. Immunol. Methods 65: 55–63, 1983.
U.S. Pat. No. 6,403,378 describes a method based on membrane integrity that utilizes two dyes, one of which labels all intact cells while the other labels all dead cells. The methodology permits all non-viable cells to be detected at one wavelength while all viable and non-viable cells can be detected at a different wavelength.
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